Behavioral reproductive isolation among sympatric strains of Brachionus plicatilis Müller 1786: insights into the status of this taxonomic species

We present results on cross-mating experiments using Brachionus plicatilis strains collected in three ponds of a coastal marsh (Torreblanca Marsh, Castellón, Spain). These strains were known to differ widely both in morphology and allozyme patterns from a previous study, where they were grouped into three genetically different clonal groups. Although some of the strains co-occurred in the same pond and sexual periods overlapped, no gene flow was found among them. Our first objective was to determine whether behavioral reproductive isolation was responsible for the absence of interbreeding. A second objective was to explore the relationship between sexual isolation and genetic divergence. We performed two experiments. In Experiment 1, we tested five strains from different clonal groups; in Experiment 2, we added a strain from a congeneric species, and strains from different ponds. We recorded male mating behavior in all possible male-female strain pairings. Our data show that males of a strain tend to mate with females of the same strain or genetically similar strains, regardless of the pond they come from. We also found a high positive correlation between isolation distance and genetic distance. These results support the view that mating behavior acts as an important isolating barrier giving cohesion to clonal groups, and structuring populations of this rotifer, and that Brachionus plicatilis is a taxon composed of more than one biological species.     

A family of modular type mannuronan C-5-epimerase genes controls alginate structure in Azotobacter vinelandii

The ToxR protein is a transmembrane protein that regulates the expression of several virulence factors of Vibrio cholerae. Previous analysis of fusion proteins between ToxR and alkaline phosphatase (ToxR-PhoA) suggested that ToxR was active as a dimer. In order to determine whether dimerization of the ToxR periplasmic domain was essential for activity, this domain was replaced by monomeric and dimeric protein domains. Surprisingly, PhoA (dimeric), β-lactamase (monomeric, ToxR–Bla), or the leucine zipper of GCN4 (dimeric, ToxR-GCN4-M) could substitute functionally for the ToxR periplasmic domain. ToxR-GCN4 fusion proteins, in which the ToxR trans-membrane domain was eliminated (ToxR-GCN4-C), were inactive, but an additional fusion protein that contained a heterologous membrane-spanning domain retained activity. Strains containing each of these ToxR fusion proteins were analysed for in vivo colonization properties and response to in vitro growth conditions that are known to affect expression of the ToxR regulon. Strains containing ToxR-GCN4-M and ToxR-Bla responded like wild-type strains to in vitro growth conditions. In the infant-mouse colonization model, strains containing ToxR fusion proteins were all deficient in colonization relative to strains containing wild-type ToxR, and strains containing monomeric ToxR-Bla were most severely outcompeted. These results suggest that, under in vitro conditions, ToxR does not require a dimerized periplasmic domain, but that, under in vivo conditions, the correct conformation of the ToxR periplasmic domain may be more important for function.     

Conformational and functional similarities between glutaredoxin and thioredoxins.

The tertiary structures of thioredoxin from Escherichia coli and bacteriophage T4 have been compared and aligned giving a common fold of 68 C alpha atoms with a root mean square difference of 2.6 A. The amino acid sequence of glutaredoxin has been aligned to those of the thioredoxins assuming that glutaredoxin has the same common fold. A model of the glutaredoxin molecule was built on a vector display using this alignment and the T4 thioredoxin tertiary structure. By comparison of the model with those of the thioredoxins, we have identified a molecular surface area on one side of the redox-active S-S bridge which we suggest is the binding area of these molecules for redox interactions with other proteins. This area comprises residues 33-34, 75-76 and 91-93 in E. coli thioredoxin; 15-16, 65-66 and 76-78 in T4 thioredoxin and 12-13, 59-60 and 69-71 in glutaredoxin. In all three molecules, this part of the surface is flat and hydrophobic. Charged groups are completely absent. In contrast, there is a cluster of charged groups on the other side of the S-S bridge which we suggest participates in the mechanisms of the redox reactions. In particular, a lysine residue close to an aromatic ring is conserved in all molecules.     

Conservation of highly repetitive DNA in cetaceans

It is controversial whether odontocetes (toothed whales) and mysticetes (whalebone whales) have a common ancestry. Cetacean karyological uniformity, which is unique among mammalian orders, suggests a monophyletic origin; however, several anatomical authorities have maintained that odontocetes and mysticetes are diphyletic. We investigated the issue using Southern blot hybridization. Two labelled restriction fragment probes from the DNA of the sei whale (a mysticete) were hybridized to restricted DNA of cetacean species representing all extant families except the Eschrichtiidae, the gray whales. The probes hybridized to specific restriction fragments in all odontocete and mysticete materials. Hybridizations showed preservation of hybridization homologies and a striking conservation of the length of highly repeated DNA sequences. The results are compatible with a common ancestry of odontocetes and mysticetes.     

Polarization and fluorescent microscopy —Simple methods for demonstrating pulmonary surfactant lipids

Summary Birefringence and fluorochrome lipid staining with benzpyrene are demonstrated as simple morphological methods to reveal the presence or absence of surfactant lipids in human newborn lung tissue. Lack of lipid birefringence proves to be an associated finding in the lungs of premature infants with hyaline membrane disease, indicating the possible pathogenetical importance of surfactant deficiency.     

The fluctuations in the level of endogenous growth regulators in seeds ofAcer pseudoplatanus L. in the course of stratification

In the course of cold stratification ofAcer pseudoplatanus L. fruits a statistically significant alternation occurs in their seeds of a rise and fall in the level of endogenous growth regulators. In the initial weeks the inhibitory effect slightly declines, or, on the contrary, the stimulatory effect slightly increases; in the middle phase of stratification a marked increase in inhibitions and reduction of stimulations appears, and towards the end of stratification the stimulatory effect of isolated substances in the individual biotests rises again, or their inhibition effect is decreased. No direct dependence was found between the decrease of the degree of dormancy and the drop of inhibitor, or increase of promotor levels. However, a certain analogy was observed between the time course of fluctuations in the level of growth regulators and the germination energy of the seeds investigated. An enhancement of the growth activity of the substances isolated (e.g. those of a gibberellin-like nature) in the last weeks of stratification can already be considered as the result of the release of fruits from dormancy.     

Molecular properties of the red cell calcium pump: II. Effects of calmodulin,proteolytic digestion and drugs on the calcium-induced membrane phosphorylation by ATP in inside-out red cell membrane vesicles

In inside-out human red cell membrane vesicles /IOV/, in the absence of Mg²⁺, the only calcium-induced labelling by γ³²P-ATP occurs in a 140–150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated intermediate /EP/ of the calcium pump. In the presence of Mg²⁺ calcium-induced phosphorylation is accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accelerates EP formation both in the absence and presence of Mg²⁺.Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVs, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of EP. In trypsin-digested IOVs the molecular weight of the ³²P-labelled EP is shifted to lower values /110–120 000/ We suggest that trypsin digestion cleaves off a 20–40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule.     

A study of the role of histidine side-chains at the active centre of amylolytic enzymes

The role of histidine side-chains in reactions catalysed by porcine-pancreatic alpha-amylase, sweet-potato beta-amylase, and Aspergillus niger glucamylase has been studied by using diethyl pyrocarbonate, a specific protein reagent. Changes in the activity, binding affinity, and apparent kinetic parameters due to ethoxycarbonylation have been determined. For pancreas alpha-amylase, four of the eight histidine groups, for sweet-potato beta-amylase, six of the seven histidine groups, and for A. niger glucamylase, four of the six histidine groups were shown to be ethoxycarbonylated. Ethoxycarbonylation occurred as an apparent first-order reaction, with rate constants in the range 3.6–4.9 x 10^?2min^?1. Ethoxycarbonylation of the histidine group at the active centre rapidly inactivated alpha-amylase, whereas the other three groups are not located in the active centre, although activity and substrate binding are only slightly affected by their modification. For beta-amylase and glucamylase, only slight or no change in activity could be detected on ethoxycarbonylation, whereas significant changes were observed in the binding affinity.     

Functionally-stabilized proteins--a review

The maintenance or stabilization of protein or enzyme function is of vital importance in Biotechnology. Investigations of thermophilic organisms, studies of denaturation and the use of enzymes in organic solvents have each contributed to an understanding of protein stability. Enzymes can reliably and reproducibly be stabilized by variety of means including immobilization, use of additives, chemical modification in solution and protein engineering. Examples of each of these are discussed. With these recent advances it appears that a rational strategy for achieving a particular stabilized enzyme or protein may be within reach.